Recent studies with monoclonal antibodies have demonstrated both increasing complexity in the antigenicity of the basement membrane zone (BMZ) of normal human skin and a possible role for the abnormal expression of one or more of these antigens in the pathogenesis of lesions in selected bullous diseases, most notably inherited epidermolysis bullosa (EB). In particular, the 19-DEJ-1 monoclonal antibody, which was produced during the first 3 years of funding of this project, has proven to be the most sensitive and accurate probe available for the diagnosis (both postnatal and prenatal) of all forms of junctional EB. During the next proposed funding period, six specific objectives will be pursued with regard to the 19-DEJ-1 antigen: (1) The antigen will be isolated and purified from human tissues (skin, placenta, other) and one or more cell lines (by chemical extraction and immunoaffinity chromatography); (2) the nature of this antigen will be more precisely characterized by a variety of biochemical, biophysical, and immunological techniques (including immunoprecipitation, one and two-dimensional PAGE, analysis of amino acid and carbohydrate composition, peptide "fingerprinting" via chemical and enzymatic degradation, rotary shadowing, and immunoblot {the latter via production of additional blottable monoclonal and polyclonal antibodies to the same antigen}); (3) precise ultrastructural localization of the antigen within skin BMZs will be achieved via immunoelectron microscopy and immunogold labeling techniques; (4) comparative semi-quantitative assays of the antigen will be performed on extracts of normal and junctional EB tissues and cultured keratinocytes; (5) one or more of the 19-DEJ-1 antigen-specific antibodies will be used to screen human placental and keratinocyte cDNA libraries which have been inserted into lambda-gt 11 bacteriophage vectors; and (6) selected in vitro studies will be performed to assess the possible interactions between 19-DEJ-I antigen, other skin BMZ components, and keratinocytes. Collectively, these studies should better define not only the biochemical composition of a novel hemidesmosome-associated component of the skin lamina lucida but more importantly, its possible role in the pathogenesis of altered mechanical stability of the epithelial-connective tissue interface and blister formation in one of the severest forms of inherited EB.